The present invention relates to polypeptides with in vitro chondrogenic and in vivo osteogenic activity.
Proteins capable of inducing bone morphogenesis (BMPs) were first identified from extracts of demineralized bone (Urist, 1965), and shortly thereafter from teeth (Bang and Urist, 1967). The bone-derived osteogenic activity was the subject of extensive isolation and purification efforts, until the human BMPs 2-7 were successfully punified and cloned (Wang et al., 1988; Wozney et al., 1988). The BMPs are members of a larger family of growth factors known as the DVR/TGF-xcex2 supergene family (Kingsley, 1994). Although the BMPs were originally studied for their osteogenic inductive capabilities, following their cloning an explosion of developmental research revealed that the BMPs have a variety of regulatory functions throughout development, and at the embryonic level, in the transfer of signals during epithelial-mesenchymal interactions.
Bones and teeth are related tissues in that the bone and dentin matrices are comprised of type I collagen fibers that become mineralized by impregnation with crystals of carbonated calcium hydroxyapatite. It can thus be argued that bone and dentin mineralization follow similar pathways, although they do differ in detail. Most of the extracellular matrix proteins that have been found in bone have also been described in dentin. However, dentin contains several specific proteins that have not yet been found in bone.
Bone normally undergoes constant remodeling, with formation and degradation carried out in parallel. Teeth, once they have reached maturity, grow only with the addition of secondary dentin at a low rate, with little if any, normal resorption. Developmental processes represent another major difference. Membrane bone and the bone from the appendicular skeleton are formed by osteoblasts originating from mesenchymal stem cells. Their differentiation and maturation are governed by numerous cytokines and para- and autocrine factors. The non-membrane bones follow an endochondral model during osteogenesis. In contrast, during tooth development, epithelial cells differentiate into ameloblasts, which secrete the enamel, while ectomesenchymal cells derived from the neural crest differentiate into odontoblasts, which secrete the dentin. These differentiation processes occur through a set of staged reciprocal interactions between the epithelial and mesenchymal cells, leading to the formation of the mature tooth (Lumsden, 1988). Epigenetic signals passed between the cells appear to induce the appropriate cellular morphogenesis events. BMP-4 has been localized within the tooth germ and has been proposed as one of the signals regulating the reciprocal transfer of information (Vainio et al., 1993), although BMP-4 alone cannot completely reproduce these changes, and other signals appear to be necessary.
Bang and Urist (1967) demonstrated that demineralized dentin matrix had the same potential as bone matrix to induce uncommitted mesenchymal cells to differentiate, at ectopic sites in vivo, into cartilage and then bone, following a pathway that resembled endochondral bone formation. In fact, dentin matrix implants were more potent in bone induction than bone matrix implants (Urist and Strates, 1971; Somerman et al., 1987; Veis et al., 1989).
By in situ hybridization, transcripts for BMPs-2 and 4 have been localized to the odontoblast cell layer in mouse embryos (Lyons et al., 1990; Vainio et al., 1993). Dentin matrix probably does contain the expressed BMPs. However, the BMP proteins themselves have not been successfully purified from dentin. Beesho et al. (1990, 1991) isolated proteins with BMP-like activity from rabbit and human dentin, but the NH2-terminal amino acid sequence for the human dentin-derived BMP did not resemble that of the known BMP family. In this laboratory, Amar et al. (1991 ) described the isolation from rat incisor dentin of a polypeptide exhibiting chondrogenic inducing activity (CIA) in cell culture assays (Koskinen et al. 1985, Veis et al., 1989). The amount of protein recovered from rat teeth was small and the NH2-terminal sequencing data was very limited. Nevertheless, as in the report of Bessho et al. (1991), it was clear that the CIA did not correspond to the known BMPs.
It is an object of the present invention to identify and characterize the dentin matrix protein exhibiting chondrogenic and or osteogenic activity.
The present invention provide chondrogenic and osteogenic inducing molecules (CIM), also called chondrogenic inducing agents (CIA).
In one aspect, the invention provides a chondrogenic and osteogenic inducing molecule having the nucleotide sequence SEQ ID NO: 16. This polynucleotide corresponds to a splice product of the rat amelogenin gene encoded by rat amelogenin gene exons 2, 3 ,4, 5, 6 and 7.
In a preferred embodiment of this aspect, the invention provides a chondrogenic and osteogenic inducing molecule, also referred to herein as xe2x80x9crA4xe2x80x9d, that has the nucleotide sequence SEQ ID NO: 1. rA4 corresponds to a specific defined splice product of the rat amelogenin gene encoded by rat amelogenin gene exons 2, 3 ,4, 5, 6d and 7.
In another aspect, the invention provides a chondrogenic and osteogenic inducing molecule that has the nucleotide sequence SEQ ID NO:17. This polynucleotide corresponds to a splice product of the rat amelogenin gene that includes exons 2, 3, 5, 6 and 7 of the rat amelogenin gene.
In a preferred embodiment of this aspect, the invention provides a chondrogenic and osteogenic inducing molecule, also referred to herein as xe2x80x9cr(A-4)xe2x80x9d that has the nucleotide sequence SEQ ID NO:2. r(A-4) corresponds to a specific defined splice product of the rat amelogenin gene that includes exons 2, 3, 5, 6d and 7 of the rat amelogenin gene.
Another aspect of the present invention is directed to nucleic acid sequences complementary to, or showing sequence similarity to, the DNA sequences identified in SEQ ID NOS: 1, 2, 16 and 17. The present invention is also directed to those sequences which are at least 60%, preferably at least 80%, and most preferably at least 95%, especially 98%, identical thereto.
These molecules have surprisingly been found to be useful to induce differentiation of cells to the osteogenic and chondrogenic phenotypes and may be used in a composite cell construct for bone and cartilage regeneration.
Accordingly, the invention provides a composition and a method for enhancing osteogenic and or chondrogenic generation or growth. The method comprises exposing cells to the chondrogenic/osteogenic polypeptides of the invention.